Our laboratory has shown that amphetamines trigger the internalization of the dopamine transporter (DAT) by a series of intracellular events that are distinct from the generally established actions of amphetamines to inhibit dopamine (DA) uptake or to increase DA efflux. We have found that when applied to cell lines, cultured DA neurons or midbrain slices, amphetamine activates the small GTPases, RhoA and Rac-1 and triggers internalization of DAT by a specialized internalization pathway that requires the activation of the small GTPase, RhoA. Intriguingly, amphetamine must be transported into the cell to have these effects and its actions are actually blocked by cocaine, a drug that inhibits DAT and prevents amphetamine entry. We have also found that elevation of cAMP, via DA receptors or by amphetamine-induced adenylate cyclase activation, inactivates RhoA and serves as a break on carrier internalization, thus demonstrating an important interaction between PKA- and RhoA-dependent signaling in mediating the actions of amphetamines. These observations also imply the existence of a novel intracellular target for amphetamines and suggest new cellular pathways to target in order to disrupt amphetamine action. We have also observed that the same amphetamine-activated RhoA-dependent mechanism also downregulates a glutamate transporter, EAAT3, present on DA neurons. We have identified the EAAT3 amino acid sequence responsible for this regulation, generated a cell-permeant fusion protein that interferes with the process and have used it explore the effects of amphetamine on excitatory neurotransmission in brain slices. These findings provide a new context in which to consider the actions of amphetamine on dopaminergic and glutamatergic signaling, and should provide insight into the unique neurotoxic and behavioral properties of this class of psychostimulant drugs. Recent work in the laboratory has established the role of a trace amine receptor (TAAR1) a G-protein coupled receptor found in dopamine neurons, which may serve as a direct intracellular target for amphetamines. Using transgenic mouse lines lacking the TAAR1 receptor we have found that some of the intracellular effects of amphetamine on dopamine neurons can be linked to TAAR receptor activation. We have also identified a number of human TAAR1 variants that display altered function and have been using transfected cell lines to characterize the impact of these mutations on amphetamine sensitivity. G-protein beta-gamma subunits that are released following the activation of G-protein-coupled receptors have been shown to directly bind and inhibit the activity of the dopamine transporter. An additional line of research in the laboratory has used cell permeable peptide fragments and mutagenesis to define the domain on the transporter required for this interaction and developed structural models for how this interaction may facilitate dopamine efflux by the transporter. Glutamate transporters (also known as excitatory amino acid transporters or EAATs) present at the surface of neurons and supporting glial cells regulate the extracellular concentration of glutamate, the major excitatory neurotransmitter in the brain. By transporting glutamate back into the cell, these carrier proteins prevent glutamate from reaching toxic levels and also limit the extent and duration of transmitter signaling during glutamatergic neurotransmission. These carriers have an additional function in that they possess an anion channel activity that can regulate cellular excitability, which enables them to serve as sensors of glutamate levels outside the cell. Our laboratory has used site-directed mutagenesis, sulfhydryl modification, and chemical cross-linking approaches together with biochemical, and electrophysiological analyses of the mammalian carriers to examine the structural domains required for substrate transport and ion permeation. We have also developed new approaches that use chemical modifications of cytoplasmic cysteine residues to capture inward-facing conformations of these transporters. This work has shown that inward facing conformations of the carriers also mediate the anion channel activity associated with glutamate transporters. Overall, these results shed light on how the structure and conformation of these transporter proteins determine their functional dynamics and regulatory properties. The neuronal glutamate transporter isoform, EAAT3 has also been shown to transport the amino acid cysteine and has been proposed to be a primary mechanism used by neurons to obtain cysteine for the synthesis of glutathione, a molecule critical for preventing oxidative stress and neuronal toxicity. This year the laboratory also completed work examining the mechanism of cysteine transport by EAAT3. These studies showed that the transport of cysteine through EAAT3 requires formation of the thiolate form of cysteine in the binding site. These studies assessed the transport kinetics of different substrates and measured transporter-associated currents electrophysiologically. Using a membrane tethered pH sensor to monitor intracellular pH changes associated with transport activity, we observed that transport of acidic substrates such as L-glutamate or L-selenocysteine by EAAT3 decreased intracellular pH, whereas transport of cysteine resulted in cytoplasmic alkalinization. Under conditions that favor release of intracellular substrates through EAAT3 we observed release of labeled intracellular glutamate but did not detect cysteine release. Our results support a model whereby cysteine transport through EAAT3 is facilitated through cysteine de-protonation and that once inside, the thiolate is rapidly re-protonated. Moreover, these findings suggest that cysteine transport is predominantly unidirectional and that reverse transport does not contribute to depletion of intracellular cysteine pools.